Plasmacytoid dendritic cells (pDCs) are professional interferon (IFN)-producing cells of the immune system3-8 that are specialized in recognizing viral RNA and DNA via endosomal TLR7 and TLR9, respectively. Among the TLRs that associate with MyD88, TLR7 and TLR9 strictly depend on MyD88 for signal transduction, and reside in the endoplasmic reticulum (ER) in association with UNC93B and gp96. Engagement of TLR9 by CpG-A oligodeoxynucleotide (ODN) in the early endosomes preferentially triggers the TRAF3/IRAK1/IKK1/PI3K/IRF7 signal cascade leading to type 1 IFN responses, whereas engagement of TLR9 by CpG-B ODN in the late endosomes preferentially triggers the TRAF6/BTK/IRAK4/TAK1/IRF5/NF-kB signal cascade leading to the production of proinflammatory cytokines TNF and IL-6. However, the molecular mechanisms underlying the specialization of TLR9-mediated early endosomal IFN responses versus late endosomal proinflammatory cytokine responses are unknown. We discovered that PACSIN1, a member of the PACSIN (protein kinase C and casein kinase substrate in neurons) family, was specifically expressed by pDCs within the immune system. Our preliminary studies showed that knockdown of PACSIN1 expression in human pDCs or knockout of the Pacsin1 gene in mouse pDCs led to a great reduction in the early endosomal IFN response to CpG-A ODN without affecting the late endosomal proinflammatory cytokine response to CpG-B ODN. Our central hypothesis is that the molecular basis for the differential TLR9-mediated early and late endosomal responses to DNA is determined by the use of different adaptor molecules. The first aim will focus on the potential role of in CpG ODN trafficking by using a human pDC cell line with stable knockdown of PACSIN1 expression by shRNA and mouse pDCs derived from PACSIN-deficient mice. The second will focus on identification of PACSIN1-binding proteins by yeast two-hybrid screening and co-IP followed by mass spectrometry. The third aim will focus on further characterization of three potential PACSIN1-interacting molecules including CD2AP, MEKK4 and TRAF4. PUBLIC HEALTH RELEVANCE: This proposal will focus on the characterization of the molecular pathways in TLR9- mediated early endosomal type 1 IFN-responses to viral DNA by plasmacytoid dendritic cells (pDCs). By combining microarray gene expression analysis, yeast two-hybrid screening, co-IP, mass spectrometry and gene targeting technology, four potential molecules including PACSIN1, CD2AP, MEKK4 and TRAF4 will be studied. Understanding pDC type 1 IFN responses to DNA will be highly significant for the development of therapies for viral infectious diseases and autoimmune disorders.